So far, biochemical assaying of selected target substances in biological sample has been widely used in association with clinical diagnosis. As a typical example of biochemical diagnosis, there may be mentioned the detection of fungi or gram-negative bacteria utilizing the Limulus reaction.
The Limulus reaction is a biochemical reaction in which a Limulus reagent is used.
In the horseshoe crab (Limulus) amebocyte lysate (hereinafter sometimes referred to as "lysate" for short) contained in the Limulus reagent, there coexist a cascade type coagulation system (factor C system), which reacts with endotoxin and is thereby activated, and a cascade type coagulation system (factor G system), which reacts with (1.fwdarw.3)-.beta.-D-glucan (hereinafter sometimes referred to as ".beta.-glucan") and is thereby activated (FIGURE 1). A method of specifically assaying endotoxin utilizing the former system alone and a method of specifically assaying .beta.-glucan utilizing the latter system alone are known [Obayashi, T. et al., Clin. Chim. Acta, 149, 55-65 (1985)]. Patients with fungal infection show increased blood .beta.-glucan levels while patients suffering from infection with gram-negative bacteria show increased blood endotoxin levels. It is also known that infections with fungi or gram-negative bacteria can be diagnosed by determining blood .beta.-glucan or endotoxin levels.
Such methods of assaying .beta.-glucan utilizing the factor G system and of assaying endotoxin utilizing the factor C system show very high detection sensitivity and therefore are suited for use in detecting both the substances mentioned above occurring in trace amounts in biological samples. In particular, their effectiveness in diagnosing deep fungal infections and infections caused by gram-negative bacteria has been studied and confirmed, and said methods are already in use to a certain extent in clinical testing.
Meanwhile, in determining .beta.-glucan or endotoxin in biological samples, in particular blood samples utilizing the cascade reaction involving the factor G or factor C system of the lysate, respectively, said reaction is based on the reaction catalyzed by serine protease in the lysate and therefore various interfering factors contained in the samples (e.g. thrombin and factor Xa, which show similarity in action to clotting enzyme in the lysate and therefore behave as false positive factors, and .alpha..sub.2 -plasmin inhibitor, .alpha..sub.1 -antitrypsin and antithrombin III, which strongly inhibit the reaction, hence are false negative factors) must be deactivated or eliminated. Pretreatment is thus required. The prior art method so far employed for this purpose comprises preparing platelet rich plasma (PRP) by subjecting a blood samples to a specific treatment, further adding perchloric acid, incubating the resulting mixture at 37.degree. C., removing the denatured product precipitate by centrifugation, collecting the supernatant and neutralizing the same with an alkali to give a test solution [Obayashi, T. et al., Clin. Chim. Acta, 149, 55-65 (1985)]. However, this method has problems. For instance, the procedure for separating the denatured product precipitate is complicated or troublesome, the whole procedure comprises a number of steps, and there is the risk of the reaction system being contaminated with a substance affecting said system during operation.
The prior art methods of pretreatment for assaying endotoxin or .beta.-glucan are invariably carried out in test tubes and portions of the samples thus pretreated are transferred to other test tubes for performing the Limulus reaction. Furthermore, in conducting the assay by the synthetic substrate method, the end point technique has been generally employed which comprises converting, following the Limulus reaction, p-nitroaniline, formed upon substrate cleavage, to a red color through the diazotization reaction and measuring the absorbance. The end point technique is generally complicated in procedure and requires a rather long measurement time. A method by which a number of samples can be treated within a short period of time is demanded. Although the use of microplates in lieu of test tubes makes it possible to handle a large number of samples at a time, continuous automatic measurement by the end point technique is difficult to perform using microplates.
A kinetic assay method so far proposed (JP-A-3-220456) which can use such microplates and can determined the change of a substrate directly and automatically is therefore a desirable assay method (the term "JP-A" used herein means an unexamined published Japanese patent application). However, accurate and precise assay is impossible since the reaction mixture volume on microplates is small and the assay is readily affected by turbidity of the reaction mixture, for instance.
Furthermore, the method of pretreating samples for endotoxin assay is different from that for .beta.-glucan assay since the reaction system to be used in the former assay is different from that to be used in the latter. If one and the same sample is to be assayed for both endotoxin and .beta.-glucan, the sample requires different methods of pretreatment; this is very laborious and uneconomical. A pretreating reagent or reagent system which can be used in assaying both and with which only one pretreatment procedure is required is earnestly desired.
If a general-purpose pretreating reagent or reagent system is available with which various biological samples can be pretreated and rendered applicable to various biochemical reactions, such reagent or reagent system will eliminate the necessity of complicated pretreatment procedures differing from one another depending on the kind of assay method and the kind of biological sample and will make a very great contribution to biochemical research, diagnosis, etc. Such a pretreating reagent or reagent system is also desired.